A 3 D model will be developed in aggregate cultures to conduct developmental neurotoxicity testing (DNT). The model will capture the complex morphological and biochemical interactions between astrocytes and neurons that take place during development. Astrocytes participate in neuronal differentiation by providing a matrix for axonal growth and dendritic arborization. They participate in synapse formation and monitor synaptic activity through their processes that ensheath synapses. Astrocytes remove glutamate from the synapse, they respond to glutamate and ATP through a calcium response, and they detoxify reactive oxygen species generated by the active mitochondria and by neurotransmitter biosynthesis. Aggregate cultures were found to promote neuronal differentiation, synapse formation, and myelination in brains cells derived from rodents and humans. In the specific aims, neuronal differentiation, synapse formation, synaptic plasticity, and responsiveness to substances will be compared between co-cultures of astrocytes and neurons in aggregate and monolayer cultures. Astrocytes and neurons will be developed from human induced pluripotent stem cell lines (iPSC). Synapse formation will be assessed morphologically. Astrocyte participation in synapse formation will be determined morphologically by measuring the juxtaposition of astrocyte processes with synapses. Functional assessment of astrocyte participation will be accomplished with genetically driven calcium sensitive fluorescent probes. Similarly, the responses of neurons and astrocytes to different substances that increase oxidative stress and inflammation will be measured with biochemical assays and reporter gene constructs containing fluorescent probes. Because genetic variability will influence responses to drugs and other substances, the DNT model will also be developed with iPSC from different genetic backgrounds. To demonstrate the feasibility that genetic background is important in DNT, iPSC from individuals with Down's Syndrome and Tuberous Sclerosis Complex will be tested. In both syndromes, the genetic modification increases sensitivity to substances that generate oxidative stress. Several DNT substances will be examined in aggregate cultures of neurons and astrocytes induced from iPSC that are derived from both syndromes. Overall, the model developed in this proposal will have the potential to assess DNT in human neurons and astrocyte derived from individuals with different genetic backgrounds.

Public Health Relevance

A 3D rat mini brain model successfully used for developmental neurotoxicity testing will be humanized using induced pluripotent stem cells. This allows to use cells from healthy donors and patients and study for the first time gene/environment interactions. The model promises to replace demanding animal testing with a more predictive human model.